Over the past two decades, a great deal has been learned about the signaling events for normal and pathologic platelet activation. Activation of members of the phosphoinositide 3-kinase (PI3K) family is an early event after platelet stimulation. Although in other hematopoietic cells PI3K has been implicated in proliferation, survival, and actin reorganization, relatively little is known about its role in platelets except that PI3K inhibitors block platelet aggregation. To date, three isoforms of PI3K have been found in human platelets, and are classified by their catalytic subunit: p110alpha, p110beta and p110gamma. PI3Kalpha and PI3Kbeta are known to associate with a regulatory subunit, p85, and are tightly linked to signaling mediated by growth factor receptors. The most recently described member of this group is a heterodimer composed of a regulatory unit called p101 and a kinase unit called p110gamma(PI3Kgamma). Unlike other phospholipid kinases, PI3Kgamma is directly stimulated by both G protein betagamma heterodimers. Therefore, PI3Kgamma represents an important new link between G-protein coupled receptors and a phospholipid signaling pathways once thought to be activated exclusively by growth factor receptors. As an extension of our work with the platelet protein plackstrin, we have found that pleckstrin in a phosphorylation-regulated fashion inhibits the PI3Kgamma while having no effect on the other PI3K isoenzymes. Using a well described PI3K inhibitor, LY294002, we have found that this enzyme is important for agonist-stimulated platelet secretion. In additional experiments using transfected nucleated cells, we demonstrated that in response to stimulation of G-protein coupled receptors, this PI3Kgamma induces actin reorganization and translocates to the nucleus. Furthermore, we have identified several mutations that enhance or disrupt these phenomenon. The studies with inhibitors suggest that PI3K plays a significant role in the signal transduction pathways that activate platelets. Our preliminary data also raise the possibility that PI3K could be involved in megakaryocyte proliferation and the unusual nuclear biology that characterizes these cells. To better understand the function of PI3k in platelets and megakaryocytes, the studies in this proposal will address the following questions: (1) What is the role of PI3K on platelet signaling? (2) Does PI3K play a role in megakaryocytes? and (3) How are the different platelet PI3K isoforms regulated?